AcyI-CoA synthetase (ACS) catalyzes the ligation of fatty acids to CoA yielding acyI-CoA, thus completing the initial step in fatty acid metabolism. Three isoforms of ACS predominate in rat liver with each isoform having different subcellular locations and substrate preferences, which suggests distinct roles of each isoform in partitioning fatty acids to distinct metabolic pathways. However, the function of each isoform in fatty acid metabolism has not be elucidated. Additionally, polyunsaturated fatty acids induce gene expression of enzymes involved in fatty acid oxidation and suppress genes involved in lipogenesis by upregulating peroxisome proliferators-activated receptor alpha and down-regulating sterol regulatory element binding protein-lc. In contrast, acyI-CoAs antagonize the actions of fatty acids on gene expression and upregulate genes encoding opposing pathways. Thus, we propose that controlling the conversion of fatty acids to acyI-CoAs and their pool size may be a critical step in regulating gene expression. Since ACS catalyzes this reaction, it is a novel target to regulate gene expression. The proposed research will use RNA interference to define the roles of ACS 1, 4 and 5 in hepatic fatty acid metabolism and gene expression.